Paramyxoviruses are globally prevalent human pathogens and include measles, mumps, respiratory syncytial, human parainfluenza, and the henipaviruses. The henipaviruses, represented by Nipah (NiV) and Hendra (HeV) viruses are the deadliest paramyxoviruses. NiV's mortality rate in humans is 40-92%, averaging 75% in the latest outbreaks. Animal-to-human and human-to-human transmission have been reported for NiV, underscoring the need for research and treatment development. NiV is thus classified as a Risk Group 4 and a Category C priority pathogen in the NIAID Research Agenda. Dissecting the mechanisms required for infection and spread will provide new targets for antivirals to block critical early steps in disease pathogenesis. Both paramyxovirus entry into mammalian cells (viral-cell membrane fusion) and syncytia formation (cell-cell membrane fusion) require membrane fusion, which necessitates the coordinated actions of two viral proteins: the attachment (HN, H, or G) and fusion (F) glycoproteins. How G/F interactions link cell receptor binding to F-triggering remains a critical knowledge gap for the paramyxoviruses, including NiV and HeV. Numerous structural and functional features of G/HN/H and F are conserved among the paramyxoviruses. G/HN/H has a receptor-binding globular (head) domain connected to its transmembrane anchor via a stalk domain. F is a trimeric class I fusion protein with canonical structural/functional features common to its class. Class I fusion proteins are synthesized as trimeric precursors that are cleaved for activation into a metastable pre-fusion conformation, poised for enabling membrane fusion. Cleavage generates a new hydrophobic N-terminal fusion peptide (FP) that is buried intra-molecularly until F-triggering and pre-hairpin intermediate (PHI) formation, when the FP is inserted into the target cell membrane. The PHI contains two helical regions (HR1 and HR2) with high propensity to bind each other to form a six-helix bundle (6HB), enabling membrane fusion. Our preliminary studies suggest a mechanism by which receptor binding causes structural changes in the NiV-G head that expose a NiV-G stalk C-terminal domain to trigger NiV-F. In Aim 1 we will determine if specific domains in the NiV-G head and stalk are necessary and sufficient for NiV-F triggering and viral entry. We uncovered three new fusion-modulatory regions in NiV-F: HR3, N1, and N4. In Aim 2 we will determine if upon NiV-G signaling, these fusion-modulatory regions destabilize the pre-fusion NiV-F conformation and/or modulate later steps in fusion. We discovered that NiV G/F dissociation is important during membrane fusion. In Aim 3 we will identify the most critical interactive domains in the pre-fusion G/F complex, and determine if these G/F interactive domains shift during NiV membrane fusion. Understanding the determinants of G/F that modulate membrane fusion can offer new targets for anti-paramyxoviral therapeutic design.